Mesenchymal stem cells as one kind of somatic stem cells is a generic term for cells having the ability to differentiate into cells belonging to mesenchymal cells such as osteoblasts (bone cells), adipocytes (fat cells), myocytes (muscle cells) and chondrocytes (cartilage cells). Mesenchymal stem cells are considered to exist in all types of mesenchymal tissues, and are expected to be applied to regenerative medicine such as the reconstruction of bones, muscles, blood vessels, and nerves.
Cells may be contaminated with bacteria, fungus, and the like during a subculture and may undergo hereditary changes. Human diploid cells and primary cultured cells can proliferate only for a limited number of division cycles, and age through a subculture. For these reasons, cells that are not used for a certain period of time are preserved by freezing in an ultra-low temperature freezer or liquid nitrogen tank, and are thawed as needed.
For the same reasons, mesenchymal stem cells or a mesenchymal cell population including mesenchymal stem cells are preserved by freezing when they are not used for a certain period of time. When some subjects require the regeneration of bone, cartilage or skin tissue and medical treatment for myocardial infarction, cerebral infarction, spinal cord damage, and the like, mesenchymal stem cells or a mesenchymal cell population including mesenchymal stem cells matching the respective subjects or cell types are thawed, and implantable therapeutic materials are prepared. The thawed cells are then implanted into the subjects. It is expected that such implantation will produce therapeutic effects such as the regeneration of tissue.
It is known that cells in a logarithmic growth phase are best suited for cryopreservation. For example, Non-Patent Literatures 1 and 2 disclose that importance is placed on the state of cells before freezing, and cells in a logarithmic growth phase are suitable for cryopreservation. A typical example of cryopreservation methods for cells harvested from tissue is a method (Non-Patent Literature 3) including the following steps: (1) harvesting a tissue from an individual, (2) chopping up the harvested tissue and dissociating cells from the tissue by using a proteolytic enzyme such as trypsin or collagenase, (3) suspending the dissociated cells in a culture medium, (4) primarily culturing the cells under a proper environment, (5) subculturing the cells through the second and third passages, and (6) recovering the cells in a logarithmic growth phase to use the cells for cryopreservation. However, such a cell cryopreservation method needs to undergo a step of culturing cells before cryopreservation. This step is complicated. In addition, in a business requiring the cryopreservation of a large amount of cells, like a business using a cell bank, the cost required for reagents and devices for culturing cells and facilities such as a CO2 incubator and space, etc., increases in proportion to the amount of cells to be cryopreserved. In addition, the time and labor needed for operations translate into high labor costs. Furthermore, such costs are reflected in end products and the like applied to final regenerative medicine. Under such circumstances, there have been demands for methods to reduce costs.
On the other hand, when tissue fragments harvested from an individual are directly frozen, the cells dissociated from the thawed tissue fragments exhibit a low viability and a low adhesion rate with respect to a culture flask and the like, resulting in difficulty in maintaining the cells as cultured cells.